Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His 6 -tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni The isoprenoid biosynthetic pathway is ubiquitous across all three kingdoms of life. Over 36,000 natural products derived from the fundamental five-carbon isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) have now been identified (3). Many of these molecules have important biological functions, including glycoprotein synthesis (dolichols), inter-and intracellular signaling (prenylated proteins and steroidal hormones), membrane structure (steroids), electron carriers during redox reactions (ubiquinones), photoprotection (carotenoids), photosynthesis (chlorophyll), and defense against predators (sesquiterpenes and pyrethrins).There are two independent biosynthetic routes to IPP and DMAPP (Fig. 1). In the mevalonate (MVA) pathway, first discovered in the late 1950s (4, 6), IPP is synthesized from mevalonic acid by the consecutive action of mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), and mevalonate diphosphate decarboxylase (DPMVA decarboxylase). The isomerization of IPP to DMAPP is a mandatory step needed to create the electrophilic allylic diphosphates needed for subsequent prenyl transfer reactions. IPP isomerase is an essential enzyme in archaea, eukaryota, and some gram-positive eubacteria, where IPP is synthesized by the MVA pathway. More recently, it was discovered that IPP and DMAPP are synthesized from D-glyceraldehyde phosphate and pyruvate by the methyl erythritol phosphate (MEP) pathway found in many eubacteria, cyanobacteria, and plant chloroplasts (18,28). In the final step of the MEP pathway, the ispH gene product synthesizes IPP and DMAPP from 4-hydroxydimethylallyl diphosphate (Fig. 1) (26). In organisms that utilize the MEP pathway, idi is not essential or does not exist (10).In 1997, Methanothermobacter thermautotrophicus was the first archaeon to have its genome fully sequenced (34). At th...

Isopentenyl diphosphate dimethylallyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein and is found in eukaryotes, while the type-2 isoform (IDI-2) is a flavoenzyme found in bacteria and completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in E. coli. Steady state kinetic studies of the enzyme indicated that FMNH2 (KM= 0.3 μM) bound before isopentenyl diphosphate (KM= 40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 Å resolution was obtained for the holo-enzyme, in the closed conformation with reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against closely sequence and structure related pathogens such as E. faecalis or S. aureus.

2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoidprecursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-Derythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.With more than 33,000 different compounds known to date, isoprenoids are among the most diverse groups of compounds found in nature (9). Isoprenoid molecules are important for a wide variety of cellular functions, including electron transport (quinones), stabilization of membranes (sterols), cell wall biosynthesis (dolichols), signaling (prenylated proteins and hormones), photosynthesis (chlorophylls), photoprotection (carotenoids), and protein synthesis (modified tRNAs). Despite a high degree of structural diversity, all isoprenoids are derived from two simple five-carbon molecules, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Although all higher isoprenoid molecules are derived from IPP and DMAPP, these fundamental building blocks are produced by two different biosynthetic pathways.In Archaea, Eukarya, and some gram-positive bacteria, IPP and DMAPP are synthesized from acetyl coenzyme A by the mevalonate (MVA) pathway (Fig. 1). The first two steps of the MVA pathway condense three molecules of acetyl coenzyme A to form 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) (24, 33). HMG-CoA is then reduced to MVA, which is converted to MVA diphosphate in two steps (5,8). IPP obtained by an ATP-dependent decarboxylation of MVA diphosphate is then isomerized to DMAPP (1, 15). The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is orthogonal to the MVA pathway ( Fig. 1) and is found in most bacteria and plant chloroplasts. The first step in the MEP pathway is the condensation of D-...

Essential isoprenoid compounds are synthesized using the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in many gram-negative bacteria, some gram-positive bacteria, some apicomplexan parasites, and plant chloroplasts. The alternative mevalonate pathway is found in archaea and eukaryotes, including cytosolic biosynthesis in plants. The existence of orthogonal essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. A system is described for identifying mutations in the MEP pathway of Salmonella enterica serovar Typhimurium. Using this system, point mutations induced by diethyl sulfate were found in the all genes of the essential MEP pathway and also in genes involved in uptake of methylerythritol. Curiously, none of the MEP pathway genes could be identified in the same parent strain by transposon mutagenesis, despite extensive searches. The results complement the biochemical and bioinformatic approaches to the elucidation of the genes involved in the MEP pathway and also identify key residues for activity in the enzymes of the pathway.It was recently discovered that bacteria synthesize isoprenoids by a pathway that differs from that found in eukaryotes. In bacteria, pyruvate and glyceraldehyde 3-phosphate are converted, through the 2-C-methyl-D-erythritol phosphate (MEP) pathway, to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This has focused recent research on the elucidation of the biosynthetic steps and genes encoding the catalytic enzymes in bacteria. To date, all synthetic steps of the MEP pathway and the genes encoding the biosynthetic enzymes have been identified. Isoprenoid biosynthesis begins with the condensation of pyruvate and glyceraldehyde 3-phosphate by 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, encoded by dxs (26). Next, in the first committed step of the pathway, DXP reductoisomerase, encoded by dxr, converts DXP to MEP (16,32). In the following three steps, a cytidyl monophosphate moiety is attached to the phosphate of MEP; the resulting diphosphodiester is phosphorylated at the C-2 hydroxyl in the methylerythritol moiety and then cyclized to form an eight-membered cyclic diphosphate diester, and these steps are catalyzed by the proteins encoded by ispD (25), ispE (18), and ispF (11), respectively. The enzyme encoded by ispG, 1-hydroxy-2-methyl-2-(E)-butenol 4-diphosphate (HDMAPP) synthase, catalyzes the ring opening and reduction of 2-Cmethyl-D-erythritol-2,4-cyclodiphosphate to yield HDMAPP (10). Finally, HDMAPP is converted to a mixture of IPP and DMAPP by HDMAPP reductase encoded by ispH (1).Mutants blocked in the MEP pathway of Salmonella spp. or Escherichia coli are expected to be lethal, since these organisms are unable to utilize exogenously supplied IPP, DMAPP, or their corresponding alcohols. To allow viability of such mutants, genes of the alternative mevalonate pathway were introduced into bacteria, either on plasmids or in the chromosome (2,5,9,15,24). Strains containing the ...